IVACS Student and Post-Doctoral Oral Presentation Competitions

First Place

Establishment & Scale-up Potential of Embryonic Insect Cells for Cell-cultivated Meat

Cultivated meat is a growing field dedicated to producing meat from cell culture to provide an alternative to large-scale animal agriculture, which is plagued with environmental, animal welfare, and human health concerns. While most research in the field focuses on “traditional” livestock species (bovine, porcine, avian), insect cells are promising cell sources for cultivated meat products. Insect cells are extremely resilient and adaptable in culture, and may be able to reach low-cost and large-scale culture more easily than “traditional” cells. The goal of this project was to establish an isolation procedure to develop a new insect cell line that is scalable, easily adaptable to animal-free media and generates sufficient biomass to assess nutritional properties. Cells were isolated from Manduca sexta embryos and subsequently adapted to single-cell suspension culture in animal-free growth media. Cell growth data is presented in shake flasks and pneumatic bioreactors and spent media analysis was performed using a Cedex metabolite analyzer to better understand cell metabolic processes. Finally, a preliminary proximate nutritional profile is reported using data from the Eurofins nutritional testing platform. The Manduca sexta non-adherent cells (MsNACs) that were developed were spontaneously immortalized and showed robust growth in animal-component free media, reaching densities over 20 million cells/mL in shake flasks. They were also scaled to 2.4 and 10-liter bioreactors and spent media analysis showed that they preferentially consumed glutamate over glutamine and glucose. Finally, nutritional analyses showed that MsNACs had comparable protein, fat, carbohydrate, and ash breakdown to USDA and FDA-approved chicken cells. This study presents a blueprint for developing, scaling, and analyzing novel insect cell lines for food. 

Sophie Letcher, Department of Biomedical Engineering & Tufts University Center for Cellular Agriculture, Tufts University, Medford, MA 02155. Abstract Presentation A-1012

Second Place

Enhancing dsRNA Delivery and Uptake in Salmonids Using Liposomes and Nanophytoglycogen

Therapeutics are most effective when they are delivered to their target intact and concentrated enough to perform their function with minimal toxicity to the host. One class of prophylactic molecules of interest is long dsRNA, which is produced by all viruses at some point during their replication and serves as a robust stimulant of the innate immune system against all viruses. Another is CpG-ODNs, which are short, pro-inflammatory DNA sequences. The use of a carrier molecule serves to mitigate many of the challenges posed to drug delivery by the host immune system, such as enzymatic degradation. In this study, simple cationic liposomes demonstrated significant toxicity in rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) and gonadal (RTG-2) cell lines, rendering them an impractical carrier choice for stimulation of the innate immune response. Antiviral trials proved the need for an alternative carrier to prevent infection by Chum Salmon Reovirus (CSV). Contrastingly, initial studies in RTG-2 cells revealed that cationic nanophytoglycogen NanodendrixTM (NDx) was considerably less toxic than the liposomes when conjugated with either dsRNA or CpG-ODNs and could therefore be applied in much higher concentrations. This nanoparticle, derived from sweet corn, is a potential candidate to reduce CSV infection in rainbow trout cell lines. Chinook salmon embryonic cells (CHSE-214) that had their dsRNA sensor, melanoma differentiation-associated protein 5 (MDA5), gene knocked out by CRISPR modification naturally lack the membrane scavenger receptors (SR-As) required to internalize extracellular nucleotides. They are being employed to examine the ability of NDx to translocate long dsRNA into the cytosol without the assistance of a dsRNA receptor. Further, these modified cells will help determine if NDx can enhance the innate immune response in cells lacking key cytosolic dsRNA sensors and act as an adjuvant for peritoneal injection vaccines in rainbow trout. Developing prophylactic drug delivery systems for antiviral therapeutics can help protect Ontario’s thriving aquaculture industry, situating Canada as a global competitor as both the incidence of aquaculture disease and food demand continue to rise.

Shayne Oberhoffner, Department of Biology, Wilfrid Laurier University, Waterloo, ON N2L 3C5, CANADA. Abstract Presentation A-1013

Third Place

Combined Psilocybin and Eugenol Synergistically Reduce Inflammation via the Gut-Liver Axis

Chronic liver disease is caused by multiple factors including gut dysbiosis-induced endotoxemia. Recently, psilocybin has demonstrated potent, sub-hallucinogenic, and synergistic anti-inflammatory effects on macrophages and within the brain, however, its effects on liver inflammation are unknown. We hypothesized psilocybin could ameliorate liver inflammation and prevent the development of liver diseases. Initially, psilocybin was combined with an array of transient receptor potential channel agonists and was applied to human small intestinal epithelial cells (HSEIC) and 3D EpiIntestinal tissues which were treated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ to induce an inflammatory response. Psilocybin and eugenol synergistically reduced cyclooxygenase (COX)-2 and interleukin (IL)-6 protein levels in HSEIC and was not cytotoxic. Utilizing 3D EpiIntestinal tissue, psilocybin and eugenol significantly reduced TNF-α, (IFN)-γ, IL-6, and IL-8 protein levels. Next, we injected C57BL/6J mice with lipopolysaccharide (LPS) or orally fed dextran sodium sulfate (DSS) to induce liver inflammation, and orally fed psilocybin (0.88 mg/kg), eugenol (17.59 mg/kg), or combinations of psilocybin and eugenol daily for two days as a pre-treatment or once after inflammation induction as a post-treatment. LPS and DSS upregulated mRNA expression of COX-2, TNF-α, IL-1β, and IL-6, while psilocybin and eugenol significantly ameliorated mRNA levels in the post-treatment. In contrast, pre-treatment did not result in significant improvement markers of liver inflammation. Histology in both LPS and DSS-induced models demonstrated an improvement in liver pathology. While both models demonstrated strong anti-inflammatory properties on the liver, our data demonstrates psilocybin and eugenol act directly on liver inflammation and through the gut-liver axis. Post-treatment with psilocybin and eugenol demonstrates strong potential in treating inflammatory liver diseases and should likely be studied further, including in the clinic.

Gregory I Robinson, Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K3M4, CANADA and Gies College of Business, University of Illinois Urbana-Champaign, Champaign, IL 61820. Abstract Presentation: A-1014

Post Doctoral Competition Winner

Towards a Better Understanding of TNFa Membrane-bound Form Properties with TNFR2 Receptor

Cytokines play crucial roles in immunity and present an attractive therapeutic potential. Many cytokines are initially expressed as membrane-bound and then cleaved to be released under the soluble form. Structural and functional differences between these two forms of cytokines are still unclear. However, the membrane-bound form exerts in a cell-to-cell context and might trigger a more potent cell signaling activation through a better engagement with receptors. TNFR2 is a Tumor Necrosis Factor Receptor Superfamily Member (TNFRSF), restricted to immune cells such as regulatory T-cells (Treg) that get activated upon TNFa cytokine binding to trigger cell survival or proliferation. It has been suggested that TNFR2 prefers the membrane-bound TNFa, and we would like to understand how the receptor discriminates between the soluble and membrane-form ligand.  The key to cryoEM studies is the preparation of samples mimicking native membrane-bound states. We want to reveal the receptor/ligand complex structures, clustering, and stoichiometry of multiple complexes to understand how this modulates cell signaling activation. There is a genuine need for an in-depth understanding of the mechanism by which TNFR2 is activated to understand its role in oncology and autoimmunity better.

Anissa Belfetmi-Stone, Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Abstract Presentation: A-1000